EP1503799B1 - Anticorps monoclonal immunogene - Google Patents

Anticorps monoclonal immunogene Download PDF

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Publication number
EP1503799B1
EP1503799B1 EP03726990A EP03726990A EP1503799B1 EP 1503799 B1 EP1503799 B1 EP 1503799B1 EP 03726990 A EP03726990 A EP 03726990A EP 03726990 A EP03726990 A EP 03726990A EP 1503799 B1 EP1503799 B1 EP 1503799B1
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Prior art keywords
antibody
epitope
tumor
antigen
sialyltn
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EP1503799A2 (fr
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Hans Loibner
Günter Waxenecker
Gottfried Himmler
Helmut Eckert
Manfred Schuster
Ralf Kircheis
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Meridian Biopharmaceuticals GmbH
Eleva GmbH
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Meridian Biopharmaceuticals GmbH
Greenovation Biotech GmbH
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001166—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001169—Tumor associated carbohydrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001169—Tumor associated carbohydrates
    • A61K39/001171—Gangliosides, e.g. GM2, GD2 or GD3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/00118—Cancer antigens from embryonic or fetal origin
    • A61K39/001182—Carcinoembryonic antigen [CEA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
    • C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
    • C07K16/4258—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig against anti-receptor Ig
    • C07K16/4266—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig against anti-receptor Ig against anti-tumor receptor Ig
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6056—Antibodies

Definitions

  • the present invention relates to monoclonal antibodies suitable for the production of tumor vaccines and to a method of immunogenizing tumor-associated antigens.
  • Cancer cells have virtually always, in addition to other physiological features that distinguish them from normal cells, an altered mode of glycosylation ( Glycoconj. J. (1997), 14: 569 ; Adv. Cancer Res. (1989), 52: 257 ; Cancer Res. (1996) 56: 5309 ).
  • TAA tumor-associated antigens
  • the cells as well as the tumor cells do not produce a uniform glycosylation, ie there are different glycoforms of complex glycan chains on a cell ( Annu. Rev. Biochem. (1988), 57: 785 ).
  • tumor-associated carbohydrate structures are the Lewis antigens that are increasingly expressed in many epithelial cancers. These include Lewis x, Lewis b, and Lewis y structures as well as sialylated Lewis x structures.
  • Other carbohydrate antigens are GloboH structures, KH1, Tn antigen, TF antigen, the alpha-1,3-galactosyl epitope ( Electrophoresis (1999), 20: 362 ; Curr. Pharmaceutical Design (2000), 6: 485 . Neoplasm (1996), 43: 285 ).
  • TAA are proteins that are particularly highly expressed on cancer cells, such as CEA, TAG-72, MUC1, folate binding protein A-33, CA125, EpCAM, HER-2 / neu, PSA, MART, etc. ( Sem. Cancer Biol. (1995), 6: 321 ).
  • Another approach to destroy tumor cells is an active vaccine that elicits an immune response against TAA. This immune response is thus directed against the corresponding tumor cells ( Ann. Med. (1999), 31:66 ; Immunobiol. (1999), 201: 1 ).
  • Carbohydrates are very small molecules and are therefore not directly recognized by the immune system. Carbohydrates and polysaccharides are generally considered to be thymus-independent antigens. Conjugation of immunologically inert carbohydrate structures with thymus-dependent antigens, such as proteins, enhance their immunogenicity. Vaccines based on tumor-associated carbohydrate structures are therefore coupled to so-called "carrier molecules" to increase immunogenicity ( Angew. Chem. Int. Ed. (2000), 39: 836 ). The carrier molecules are often proteins such as bovine serum albumin or KLH (keyhole limpet hemocyanin). The protein stimulates the carrier-specific T helper cells, which then play a role in the induction of anti-carbohydrate antibody synthesis ( Contrib. Microbiol. Immunol. (1989), 10:18 ).
  • both the carbohydrate antigens and the protein antigens on healthy tissues are present and are therefore recognized by the immune system as an autologous material - thus there is usually no immune response against these endogenous molecules.
  • Tumor-associated proteins are weakly immunogenic, but are still considered vaccines ( Ann. Med. (1999), 31:66 ; Cancer Immunol. Immunother. (2000), 49: 123 ; U.S. Patent 5,999,423 ).
  • One way to circumvent "self-recognition" of certain TAA is to use anti-idiotypic antibodies as immunogen that mimic the structure of the TAA, thus eliciting an immune response that also reacts with the TAA ( Cancer Immunol.Immunother. (2000), 49: 133 ).
  • the object of the present invention is to avoid the disadvantages of the tumor vaccines described in the prior art and accordingly to provide an improved tumor vaccine which produces an efficient immune response against tumor cells.
  • immunogenic means all structures that lead to an immune response in a specific host system.
  • a murine antibody or fragments of this antibody are highly immunogenic in the human organism, an effect that can be exacerbated by the combination with adjuvants.
  • An immunogenic antibody may be immunogenic by virtue of its specificity or structure.
  • the immunogenic antibody according to the invention may preferably also induce immunogenicity in the denatured state or as a conjugate with selected structures or carriers.
  • epitope defines any region within a molecule that is recognized by a specific antibody or which induces the formation of these specific antibodies. Epitopes can be conformational epitopes or linear epitopes.
  • the epitopes mimic or comprise domains of a natural, homologous or derivatized TAA. These are comparable to the TAA at least through their primary structure and possibly secondary structure.
  • the epitopes can also be completely different from the TAA in that, and purely by the similarity of spatial (tertiary) structures components of a TAA, especially proteinaceous or carbohydrate antigens mimic. Only the tertiary structure of a molecule can thus be a mimic ("immunological imitation", such as in the WO 00/73430 disclosed) which elicits the immune response against a particular TAA.
  • an antigen which mimics a proteinaceous epitope of a tumor-associated antigen is to be understood as meaning a polypeptide of at least five amino acids.
  • epitopes of the antibody of the invention is preferably at least one epitope of an antigen selected from the group of peptides or proteins, in particular EpCAM, NCAM, CEA and T-cell peptides, which are preferably derived from tumor-associated antigens, further the carbohydrates, in particular Lewis Y, sialylTn, Globo H, and the glycolipids, especially GD2, GD3 and GM2.
  • Preferred epitopes are derived from antigens specific for epithelial tumors, such as those found more frequently in breast, stomach and intestinal, prostate, pancreatic, ovarian, and lung.
  • the immunogenic antibody of the present invention may also preferentially induce a T cell-specific immune response, whereby not only antibodies of, for example, the IgM class but also the IgG class are generated in response to the administration of the antibody.
  • antigens may be used as epitopes in the sense of the invention, which generate a T-cell-specific immune response.
  • antigens especially intracellular structures or T-cell peptides can be found.
  • TAG-72 eg TAG-72, MUC1, folate binding protein A-33, CA125, HER-2 / neu, EGF receptors, PSA, MART etc.
  • TAG-72 eg TAG-72, MUC1, folate binding protein A-33, CA125, HER-2 / neu, EGF receptors, PSA, MART etc.
  • T cell epitope peptides Cancer Metastasis Rev. 18 (1999), 143 ; Curr. Opin. Biotechnol. 8 (1997), 442 ; Curr. Opin. Immunol. 8 (1996), 651
  • mimotopes of such T-cell epitopes Curr. Opin. Immunol. 11 (1999), 219 ; Nat.
  • Suitable epitopes are expressed in at least 20%, preferably at least 30% of the cases of tumor cells of a particular type of cancer, more preferably in at least 40%, especially in at least 50% of the patients.
  • Carbohydrate epitopes preferred according to the invention are tumor-associated carbohydrate structures, such as the Lewis antigens, e.g. Lewis x, Lewis b, and Lewis y structures, as well as sialylated Lewis x structures.
  • the Lewis antigens e.g. Lewis x, Lewis b, and Lewis y structures
  • sialylated Lewis x structures e.g. Lewis x, Lewis b, and Lewis y structures
  • GloboH structures, KH1, Tn antigen, particularly preferably sialylTn antigen, TF antigen, the alpha-1-3, galactosyl epitope are also preferred carbohydrate antigen structures in the context of the present invention.
  • an adhesion protein such as a homophilic cellular membrane protein, such as EpCAM
  • an adhesion protein such as a homophilic cellular membrane protein, such as EpCAM
  • active immunization can generate a variety of antibodies with specificity for the same molecule but different EpCAM binding sites.
  • the antibody of the invention is a glycosylated antibody, which glycosylation itself may also mimic an epitope of a carbohydrate epitope of a TAA.
  • Epitopes provided or imitated, wherein at least one epitope from the group of peptides or proteins and at least one epitope from the group of carbohydrates derived. It has been found that an epitope of an EpCAM protein and an epitope of a carbohydrate moiety, such as Lewis Y, or sialylTn, are preferably combined.
  • a Lewis Y-glycosylated antibody having specificity for an EpCAM structure is a particularly good immunogen in a vaccine formulation. This antibody is particularly good at mimicking cellular tumor antigens and, accordingly, effects the desired immune response to inhibit epithelial tumor cells.
  • the immunogenic antibody according to the invention preferably acts as an antigen carrier, for example a proteinaceous antigen, in the vaccine. That is, the antibody according to the invention is a multivalent antigen, for example a bi-, tri- or polyvalent antigen.
  • the epitopes are presented so that the vaccine elicits an immune response against these epitopes.
  • a vaccine is provided which contains an antibody as a di-, tri- or polyvalent antigen.
  • the antibody according to the invention is used predominantly for active immunization, and therefore administered only in small amounts. For example, no particular side effects are expected, even if the antibody of the invention is derived from a non-human species, such as a murine antibody. However, it is believed that a recombinant, chimeric, humanized or human antibody combined with murine and human components is particularly tolerated for human administration. On the other hand, a murine component in the antibody according to the invention, due to its strangeness, can additionally provoke the immune response in humans.
  • Preferred primary function of the immunogenic antibody of the invention is to present the epitopes.
  • the specific recognition of the tumor-associated antigen or the tumor-associated antigens whose epitopes it has is not absolutely necessary, but it can additionally specifically bind to an epitope and simultaneously present an epitope.
  • an antibody of the invention may be derived from a native antibody which may have been isolated from an organism or patient
  • an antibody derivative is preferably used, preferably from the group of antibody fragments, conjugates or homologues, but also complexes and Adsorbate is selected.
  • the antibody derivative contains at least parts of the Fab fragment, preferably together with at least parts of the F (ab ') 2 fragment, and / or parts of the hinge region and / or the Fc part of a lambda or kappa antibody.
  • the antibody of the invention is preferably of the immunoglobulin type, such as IgG, IgM or IgA.
  • the antibodies according to the invention may additionally contain other substances covalently in the molecular structure, such as peptides, glycopeptides, carbohydrates, lipids or nucleic acids, but also ionic groups, such as phosphate groups, or carrier molecules, such as polyethylene glycol or KLH. These side groups may themselves represent epitopes of a tumor-associated antigen within the meaning of the present invention.
  • anti-idiotypic antibodies are used for active immunization.
  • These antibodies can be provided with additional sequences or structures to obtain an immunogen according to the invention.
  • Anti-idiotypic antibodies according to the invention preferably recognize again the idiotype of an antibody which is directed against a TAA.
  • an epitope of a TAA is already formed on the paratope of the anti-idiotypic antibody as a facial expressions for the TAA.
  • the selection of epitopes is preferably made from the above-mentioned groups of TAA.
  • an anti-idiotypic antibody against glycan-specific antibodies is used, for example an anti-idiotypic antibody which recognizes the idiotype of an anti-Lewis Y antibody, for example as described in US Pat EP 0 644 947 described.
  • the immunogenic antibody according to the invention is particularly suitable as a basis for pharmaceutical preparations, in particular of vaccines.
  • pharmaceutical preparations containing a pharmaceutically acceptable carrier includes, for example, adjuvants, buffers, salts, preservatives.
  • the pharmaceutical preparations can, for. B. for the prophylaxis and treatment of cancer-associated disease states, such as the metastasis of cancer patients are used.
  • antigen-presenting cells are specifically modulated in vivo or ex vivo in order to generate the immune response against the TAA having the immunogenic antibody.
  • a preferred vaccine formulation according to the invention contains the immunogenic antibody usually only in low concentrations, for example in an immunogenic amount in the range of 0.01 ⁇ g to 10 mg.
  • the appropriate immunogenic dose is selected, for example in the range of 0.01 ⁇ g to 750 ⁇ g, preferably 100 ⁇ g to 500 ⁇ g.
  • a depot vaccine to be delivered to the organism over a longer period of time may also contain much higher levels of antibody, such as at least 1 mg to more than 10 mg.
  • the concentration depends on the amount of liquid or suspended vaccine administered.
  • a vaccine is usually provided in pre-filled syringes with a volume of 0.01 to 1 ml, preferably 0.1 to 0.75 ml. These are therefore concentrated solutions or suspensions.
  • the immunogenic antibody is preferably presented in the vaccine of the invention in a pharmaceutically acceptable carrier suitable for subcutaneous, intramuscular, but also intradermal or transdermal administration. Another mode of administration is via the mucosal route, such as vaccination by nasal or peroral administration.
  • a pharmaceutically acceptable carrier suitable for subcutaneous, intramuscular, but also intradermal or transdermal administration.
  • Another mode of administration is via the mucosal route, such as vaccination by nasal or peroral administration.
  • solids are used as adjuvants for the vaccine formulation, about an adsorbate or a suspended mixture of the antibody with the adjuvants is administered.
  • the vaccine is presented as a solution or liquid vaccine in an aqueous solvent.
  • vaccines of the tumor vaccines are already provided in a suitable pre-filled syringe. Since an antibody is relatively stable in comparison to the TAA, the vaccine according to the invention has the significant advantage that it can already be marketed as a storage-stable solution or suspension in a ready-to-use form. While a level of preservative such as thimerosal or other preservatives with improved compatibility is not necessarily required, it may be provided for extended shelf life at storage temperatures from refrigerator temperatures to room temperature in the formulation.
  • the vaccine according to the invention can also be provided in frozen or lyophilized form, which can be thawed or reconstituted if necessary.
  • vaccine adjuvants of, for example, aluminum hydroxide (Alu gel) or phosphate, eg growth factors, lymphokines, cytokines, such as IL-2, IL-12, GM-CSF, gamma interferon, or complement factors, such as C3d, liposome preparations or lipopolysaccharide from E.
  • adjuvants of, for example, aluminum hydroxide (Alu gel) or phosphate, eg growth factors, lymphokines, cytokines, such as IL-2, IL-12, GM-CSF, gamma interferon, or complement factors, such as C3d, liposome preparations or lipopolysaccharide from E.
  • LPS lipoprotein containing apoptosis factor
  • additional antigens against which the immune system has already made a strong immune response, such as tetanus toxoid, bacterial toxins, such as Pseudomonas exotoxins and derivatives of lipid A.
  • additional antigens against which the immune system has already made a strong immune response, such as tetanus toxoid, bacterial toxins, such as Pseudomonas exotoxins and derivatives of lipid A.
  • vaccine according to the invention contain further vaccine antigens, in particular anti-idiotypic antibodies, ie mixtures of the immunogenic antibody according to the invention with various antibodies which are administered simultaneously.
  • the immunogenic antibody according to the invention is also suitable for the production of diagnostic agents according to the invention.
  • reagents containing the immunogenic antibody can be offered together with other reactants or detection agents as a diagnostic agent in the form of a kit.
  • agent preferably contains a label for immediate detection of the antibody or its reaction product.
  • the diagnostic agent according to the invention is used, for example, for the qualitative and / or quantitative determination of tumor cells or metastases or for the determination of a metastasis-forming potential, wherein the agent acts by an immune reaction or immune complex formation.
  • the antibody which is assumed to be in accordance with the invention, is an anti-idiotypic antibody, ie an ab2.
  • the coupling corresponds to a conjugation to form a covalent bond. This synthesizes a derivative that is different from native antibodies.
  • TAA mimics surprisingly allows extremely efficient immunization against tumor-associated or tumor-specific structures, so that the body's immune system can be efficiently protected against the respective tumors or fight against these tumors can.
  • the antibody of the present invention functions as a proteinaceous antigen carrier present, for example, with a carbohydrate antigen as a conjugate of the present invention.
  • a carbohydrate antigen as a conjugate of the present invention.
  • carbohydrate antigens in the conjugate according to the invention.
  • several antibodies may be coupled to an antibody to elicit immune responses to two or more different tumor-associated carbohydrate structures.
  • Such a conjugate does not occur in natural systems.
  • the autoantigenic structures are thereby recognized as alien, which further enhances immunogenicity.
  • such a conjugate is present in a synthetic arrangement that is neither sterically nor functionally natural (i.e., in tumor cells).
  • low-molecular epitopes of the antigens are used for this purpose, which alone are barely recognized by the immune system of the mammals, in particular of humans.
  • the immunogenization is carried out in such a way that an antigen is conjugated to an antibody, the antibody acting as a carrier.
  • a plurality of epitopes can be immunogenic, especially naturally the epitopes of the already mentioned selection of antigens.
  • the immunogenic antibody produced preferably contains the epitope to be immunized and another epitope of a tumor-associated antigen.
  • the immunogenization yields a material that is surprisingly suitable for immunizing patients.
  • the product obtainable according to the invention is therefore preferably made available as a vaccine.
  • the carbohydrate structures chosen as epitope mimic can originate from natural or synthetic sources, the carbohydrates being present as glycoproteins or as glycolipids and as such being able to be coupled to the corresponding carrier molecule.
  • the antibody constituents can also be synthesized chemically and then combined with epitope structures or synthesized together.
  • chemical synthesis of antibody carrier molecules it is possible to introduce reactive groups at particular sites to control both the extent of coupling with an epitope and the type and location of binding.
  • the antibody carriers can also be genetically engineered as recombinant molecules. It is conceivable to produce these antibodies in host cells that do not undergo glycosylation (such as Escherichia coli). Such polypeptides may then be coupled chemically or enzymatically with a desired carbohydrate antigen.
  • the antibody carrier is produced in cells which can glycosylate the molecule.
  • the genetic modification of nucleic acids which code for native antibodies can, for example, cause corresponding glycosylation sites to be formed in the translated molecule.
  • Glycosylation of such a recombinant gene product with the corresponding tumor-associated glycan structures may be by production into cells genetically engineered to glycosylate proteins accordingly.
  • Such cells may be natural isolates (cell clones) which can be found by appropriate screening for the desired glycosylation.
  • the various epitope structures can be linked to one another via a coupler.
  • This coupler is preferably a short bifunctional molecule such as N-hydroxysuccinimide. Coupling via nitrophenyl-activated sugars is also possible. In a preferred embodiment, the coupling takes place via sulfhydryl groups ( Biochim. Biophys. Acta (1983), 761, 152-162 ). Examples of sulfhydryl-reactive linkers are BMH, DFDNB or DPDPB.
  • the coupler can also be realized by a larger chemical compound than a simple coupler molecule.
  • a coupler can also be obtained by the chemical transformation of a part of the antibody or of the antibody to be conjugated Structure can be produced quasi "in situ". This coupler produced on the antibody or on the epitope structure itself can then be conjugated directly to the other binding partner (eg via the amine group of lysine, via OH groups, sulfur groups, etc.). Coupling methods are known from the prior art ( Anal. Biochem, (1986) 156, 220-222 ; Proc. Natl. Acad. Sci., (1981), 78, 2086-2089 ; Biochem.Biophys.Res. Comm. (1983), 115, 29-37 ).
  • the antibody according to the invention comprises a nucleic acid molecule which codes for a proteinaceous TAA as an epitope structure in the context of the present invention, wherein the nucleic acid is covalently conjugated.
  • kits suitable for tumor vaccination comprises a preparation of an immunogenic antibody of the invention and a suitable application device, e.g. Syringes, infusion devices, etc. If the conjugate preparation is in lyophilized form, the kit will further contain a suitable reconstitution solution, optionally with specific stabilizers or reconstitution accelerators.
  • the immunogenic antibody having several different epitope structures in particular with the structure of a tumor-associated carbohydrate antigen, is provided, it is possible to elicit an immune response having two or more specificities and thereby combats a tumor cell with two or more different tumor-associated antigens. This widens the range of action of the vaccine and makes it more specific.
  • N-hydroxysuccinimide-activated Lewis Y tetrasaccharide is dissolved in N, N-dimethylformamide (100 mg / ml) and added dropwise to the HE2 antibody solution in appropriate buffer (100 mM sodium phosphate buffer with 150 mM NaCl, pH 8.5) and shaken for at least 2.5 hours at 4 ° C.
  • appropriate buffer 100 mM sodium phosphate buffer with 150 mM NaCl, pH 8.5
  • the extent of glycosylation of the antibody with Lewis Y can be controlled by the choice of the molar excess of activated sugar and the concentration of the antibody-containing solution (1-10 mg / ml).
  • the bispecificity of the neoglycoprotein can be detected by various immunological methods (ELISA or Western blotting with antibodies directed against the Lewis Y determinant or against HE2).
  • HE2, HE2 Lewis Y neoglycoprotein or LeY-PM (polyacrylamide-coupled tetrasaccharide, syntesome 045-PA) is dissolved in coating buffer (15 mM Na 2 CO 3 , 5 mM NaHCO 3 , 3 mM NaN 3 , pH 9.6) ( 10 ⁇ g / ml) and bound to a microtiter plate (Nunc, Denmark, Maxisorb) (1 h at 37 ° C., 100 ⁇ l / well).
  • washing buffer 2% NaCl, 0.2% Triton X-100 in PBS, 200 ⁇ l
  • 5% fetal calf serum in PBS 138 mM NaCl, 1.5 mM KOH, 2.7 mM KCl , 6.5 mM Na 2 HPO 4 pH 7.2; 200 ⁇ l
  • PBS washing buffer
  • specific anti-Lewis Y antibody human
  • goat anti-HE2 Antibody 1 ⁇ g / ml dissolved in dilution buffer: 2% FCS in PBS, 100 ⁇ l
  • the bound antibodies are amplified using HRP conjugate specific for the respective detection antibody (goat anti-human IgG + A + M HRP from Zymed (USA) for anti-Lewis Y antibody; mouse anti-goat IgG HRP (Axell , USA) for anti-HE2 antibody, 1 ⁇ g / ml, 100 ⁇ l) (30 minutes at 37 ° C).
  • HRP conjugate stains 100 ⁇ l of orthophenylenediamine dihydrochloride solution (Sigma, USA; dissolved in staining buffer and activates it H 2 O 2 ; 30%, Merck, Germany ) and the color development is stopped with 15% sulfuric acid (50 ⁇ l).
  • the absorbance developed is measured at 492 nm, the reference wavelength being 620 nm.
  • neoglycoproteins have both specificities (HE2 and Lewis Y), neoglycoprotein II is more functionally glycosylated than neoglycoprotein I and therefore gives a higher signal with the anti-Lewis Y antibody.
  • Human anti-Lewis Y antibody (10 ⁇ g / ml dissolved in coating buffer, 100 ⁇ l) is bound nonspecifically to a microtiter plate (Nunc, Maxisorb) (incubation at 37 ° C. for 1 hour), washed three times with washing buffer (200 ⁇ l ) with 5% fetal calf serum in PBS (200 ⁇ l) (incubate at 37 ° C. for 30 minutes) and with HE2-Lewis Y-neoglycoproteins I and II and HE2 as control in various concentrations (1.25-7.63x10 -6 ⁇ g / ml; 100 ⁇ l) for 1 hour at 37 ° C.
  • goat anti-HE2 antibody (1 ⁇ g / ml in dilution buffer, 100 ⁇ l) is incubated at 37 ° C for 30 minutes. Excessive antibodies are removed by a subsequent washing step (3x with washing buffer).
  • Bound antibodies are detected by incubation (30 minutes, 37 ° C) with mouse anti-goat IgG HRP (Axell, 1: 1000 dissolved in dilution buffer, 100 ul): After subsequent washing (3x with wash buffer, 1x with staining buffer) dissolves bound HRP Conjugate a staining reaction of 100 ul of added orthophenylenediamine dihydrochloride solution (Sigma, 10 mg dissolved in 25 ml staining buffer and activated with 10 ul H 2 O 2 ; 30%, Merck). The color reaction is stopped with 50 ⁇ l of 15% H 2 SO 4 and the absorbance at 492 nm (reference wavelength 620 nm) is measured on a microplate photometer (Labsystem, Model No. 354).
  • FIG. 2 it can be seen that both neo-glycoproteins can be detected in this sandwich ELISA, "Neoglycoprotein II" is more glycosylated, therefore it is more strongly retained by the pre-coated anti-Lewis Y antibody.
  • the samples are heat-treated in reducing buffer (85 ° C, 2 minutes) and electrophoresed on a polyacrylamide gel (4-12% Bis-Tris gel).
  • the size-separated proteins are visualized by silver staining (NOVEX SDS-PAGE system, Invitrogen, USA). On the gel is only a very slight increase in molecular weight due to glycosylation with Lewis Y tetrasaccharide noticeable (see FIG. 3 ).
  • the samples are separated by size. Subsequently, the separated proteins are blotted onto a nitrocellulose membrane, blocked for one hour in 3% milk powder solution and then incubated for two hours with human anti-Lewis Y antibody (10 ug / ml in PBS). Bound antibodies are detected with goat anti-human HRP conjugate (1: 500 in PBS) which is specific for the anti-Lewis Y antibody. A subsequent staining reaction visualizes the Lewis Y-glycosylated proteins.
  • neoglycoprotein II reacts with the anti-Lewis Y antibody, neo-glycoprotein I appears to be too weakly glycosylated to be detected in this assay with anti-Lewis Y.
  • Sera from immunized monkeys are screened at various times before and after immunization for the generation of a humoral immune response to HE2 and Lewis Y.
  • the immunization scheme is as follows (the individual immunizations were carried out subcutaneously: 500 ⁇ g of protein adsorbed on 1.67 mg of aluminum hydroxide in 0.5 ml of 1 mM Phosphate buffer pH 6.0 / 155 mM NaCl).
  • HE2 antibody solution is diluted to 10 ⁇ g / ml in coating buffer and incubated for 1 hour at 37 ° C (100 ⁇ l). After washing three times with washing buffer, it is blocked with 200 ⁇ l of 5% fetal calf serum in PBS for 30 minutes at 37 ° C. After a further washing step (as before), 100 .mu.l of different dilutions of the sera of immunized animals are applied to the microtiter plate (dilution buffer: 2% fetal calf serum in PBS) and incubated for 1 hour at 37.degree.
  • FIG. 5 shows the result of the HE2 ELISA. It can be seen that the immune response to the carrier protein is already very strong after 2 immunizations.
  • Lewis Y-PAA (Lectinity Holding, Inc. Bad Homburg, Germany) is diluted to 10 ⁇ g / ml in coating buffer and incubated for 1 hour at 37 ° C (100 ⁇ l). After washing three times with washing buffer, it is blocked with 200 ⁇ l of 5% fetal calf serum in PBS for 30 minutes at 37 ° C. After a further washing step (as before), 100 ⁇ l of different dilutions of the serums of immunized animals are applied to the microtiter plate (dilution buffer: 2% fetal calf serum in PBS) and incubated for 1 hour at 37.degree.
  • FIG. 6 shows that the immunization of a rhesus monkey with neoglycoprotein induces a specific Lewis Y-directed humoral immune response.
  • SialylTn-O (CH 2 ) 3 NH (CH 2 ) 4 COO pNp was coupled to HE2.
  • the final product was analyzed by SEC, LDS-PAGE, Western Blot and various ELISA assays.
  • the concentrations of HE2 sialylTn were quantified by size exclusion chromatography (SEC) on a ZORBAX GF-250 column in a Dionex system.
  • SEC size exclusion chromatography
  • the HPLC system was tested with a gel filtration standard. (BioRAD).
  • HE2 was chosen as reference standard for the quantification of HE2 sialylTn.
  • the decrease in retention time correlates with the effectiveness of the coupling reaction of sialylTn to HE2.
  • the data obtained show that the coupling efficiency increases with the reaction time and reaches saturation at 23-27 hours.
  • Fig. 7 Compared to HE2 (lanes 2-5), the HE2-sialylTn coupling product (lanes 6-8) showed a marked increase in the molecular weight of the heavy chain, suggesting that sialylTn was successfully coupled to the heavy chain (50 kDa) of the HE2 antibody has been. In addition, the appearance of a second band (of slightly different molecular weight) to the 25 kDa band indicates that also the light chain was partially coupled to sialylTn.
  • the increase in molecular weight of the HE2 antibody heavy chain after coupling with sialylTn was confirmed by Western blotting and staining with a rabbit anti mouse IgG2a-HRP.
  • a standard ELISA was performed to show how much of the anti-idiotypic binding activity (of the HE2) is retained in the coupling product.
  • Immobilized IGN111 binds the anti-idiotypic HE2 recognized by an anti-mouse IgG2a HRP.
  • HE2 is about 2-3 times more reactive than HE2 sialylTn, which means that only a slight loss of binding capacity occurs after coupling.
  • sialylTn Another standard ELISA was performed to detect sialylTn by a mouse anti-sialylTn antibody.
  • the starting material HE2 and the coupling product HE2-sialylTn were immobilized.
  • sialylTn anti-sialylTn mouse IgG
  • rat anti mouse IgG1-HRP were used.
  • SialylTn was successfully coupled to the HE2 antibody.
  • the coupling reaction has a prolonged reaction kinetics time, saturation being reached after about 24 hours.
  • SialylTn was predominantly coupled to the HE2 antibody heavy chain, whereas the light chain was only partially coupled to sialylTn.
  • the HE2-sialylTn coupling product retains most of the idiotypic specificity of HE2, the sialylTn portion of this neoglycoprotein is recognized by sialylTn-specific antibodies.
  • Example 3 Formulation of HE2 sialylTn using various adjuvants:
  • Example 4 Results of the immunization of rhesus monkeys with the HE2 sialylTn neoglycoprotein: tolerance and immunogenicity studies
  • Rhesus monkeys were vaccinated four times by subcutaneous immunizations with 500 ⁇ l of the vaccine (containing 500 ⁇ g of HE2 adsorbed on Alhydrogel in 1 mM Na phosphate buffer, pH 6.0, supplemented with 0.86% NaCl).
  • the blood samples were taken on days -3, 1, 8, 15, 29, 57 and 71.
  • the blood was allowed to clot (SST clotactivator tube (Vacutainer)); Serum was transferred to Nunc tubes 1.8 ml (375418); Day date immunization blood collection -3 No Yes 1 500 .mu.l yes, in front of the Imm. 8th No Yes 15 500 .mu.l yes, in front of the Imm. 29 500 .mu.l yes, in front of the Imm. 57 500 .mu.l yes, in front of the Imm. 71 Yes
  • Fig. 8 shows the results of immunization studies in rhesus monkeys.
  • the induction of the immune response against HE2 by the HE2-sialylTn multi-epitope vaccine is comparable to the immune response induced by HE2.
  • Pre-serum (Day 1) and immune serum (Day 15, 29, 57, 71) were evaluated for immune response against the immunizing antigen (HE2) analyzed by a HE2 ELISA and SialylTn ELISA.
  • HE2 ELISA immunizing antigen
  • SialylTn ELISA SialylTn ELISA.
  • the SialylTn ELISA was similar to the LewisY ELISA, except for a few modifications.
  • ELISA plates F96 Maxisorp microtiter plates, NUNC
  • 20 ⁇ g / ml sialylTn-PAA (30% mol, syntesome) diluted in coating buffer for 2 hours at 37 ° C.
  • the ELISA plates were blocked with 5% FCS in PBS (30 min, 37 ° C), followed by a next wash step.
  • the samples prediluted in 2% FCS
  • NAS NA pool 25-07-01, Biotest
  • a mouse anti-sialylTn CD175s antibody (DAKO, code no. M0899, lot no. 039 (601) with a starting concentration of 20 ⁇ g / ml was used, which served as a positive control.
  • the plates were stained with a goat anti-human Ig (H + L) -HRP conjugate (1: 4000, SB, Southern Biotechnology Cat.No. 2010-05, Lot No. L262-S496L) or a mouse anti-human IgG (Fc) -HRP conjugate (1: 1000, SB, Cat # 9040-05, Lot # J560-NC21G) or a mouse anti-human IgM-HRP conjugate (1: 1000, SB, Cat # 9020-05, lot # H018-W089) at 37 ° C for 30 min.
  • a rabbit anti-mouse IgG1-HRP Zymed, No. 61-0120, Lot. No. 00761146) was used.
  • the substrate OPD (1 OPD tablet dissolved in 25 ml staining buffer, + 10 ⁇ l 30% H 2 O 2 ) was added. After 10min. the dyeing reaction was stopped by addition of 50 ⁇ l H 2 SO 4 (30%).
  • Figure 9 shows the results of the sialylTn-PAA ELISA.
  • the induction of the immune response against the immunizing antigen HE2 and the target antigen EpCAM is comparable to the original HE2 single epitope vaccine.
  • there was an induction of the immune response against the carbohydrate antigen sialylTn this immune response was not induced upon vaccination by the HE2 single epitope vaccine.
  • Affinity chromatography was performed on an ⁇ KTA Explorer, Pharmacia FPLC system.
  • the diluted serum was applied to the chromatography column at a flow rate of 1 ml / min. Unbound sample was washed down with a buffer A until the UV line (280nm) was below 5mAU.
  • the desired fractions were immediately neutralized with 1M NaHCO 3 and stabilized by the addition of sodium azide (final concentration: 0.02%).
  • Purification of the pre- or immune serum was performed either by a) single-step affinity chromatography, or b) sequential affinity chromatography with the eluate of the first column applied to a second affinity chromatography column or c) differential affinity chromatography with the passage of the first Column which was loaded on a second affinity chromatography.
  • IgG immunoglobulins
  • IgM immunoglobulins
  • WM9, SKBR5, KATOIII, HT29, and OVCAR3 cells were cultured in RPMI1640 medium supplemented with L-glutamine with 10% FCS and 1% penicillin / streptomycin.
  • CT26 and CT26-KSA (clone # 21 Sp1-3; EpCAM transfected CT26 cells) were supplemented in DMEM with 10% FCS, 1% nonessential amino acids, 1% sodium pyruvate, 1% vitamin, L-glutamine and 1% penicillin / Grow streptomycin.
  • the cells were washed twice in FACS buffer and incubated with the detection antibodies under light protection for 30 minutes (sheep anti-human IgGAM-FITC (gamma and light chain specific), Silenius, dilution 1: 1000 or rabbit anti-mouse IgGAM F (ab ) 2 '-FITC Dako (gamma and light chain specific), Dilution 1: 100, for the detection of murine HE2 and KS1 / 4 antibodies. After washing three times in FACS buffer, the fluorescence intensities (10,000 cells in 100 ⁇ l FACS buffer per analysis) were measured with a FACS-Calibur system (Becton Dickinson).
  • FACS-Calibur system Becton Dickinson
  • Pre-serum (day 1) or immune serum (day 15, 29, 57, 71) of all animals was analyzed by HE2 ELISA for the immune response against the immunizing antigen (HE2).
  • HE2 ELISA immunizing antigen
  • a marked immunization effect was found in all vaccinated groups, with the strength of the immune response increasing as a function of time (and the number of immunizations).
  • none of the adjuvants increased the HE2 titer or increased the kinetics of the immune response compared to the control group receiving the antigen without adjuvant (P6 / 01).
  • the multi-epitope HE2-sialylTn vaccine (P2 / 01) induced a HE2 titer comparable to that of the HE2 vaccine (P6 / 01).
  • the HE2-sialylTn multi-epitope vaccine induces an immune response against the second antigen, sialylTn, in all immunized animals, as detected in the sialylTn ELISA, an effect not found after vaccination with the HE2 single-epitope vaccine.
  • Immune sera (day 71) were analyzed by direct EpCAM affinity chromatography followed by size exclusion chromatography (SEC). In all vaccinated groups significant amounts of Ig (IgG and IgM) were found in the immune serum (60-87 ⁇ g Ig), which was significantly higher compared to Ig content in the preserum (13-22 ⁇ g). Furthermore, an IgG switch was seen after vaccination, with elevated IgG / IgM ratios in the immune serum. In contrast, the adjuvants did not increase Ig (IgG, IgM), which have a specific reactivity with rEpCAM in the immune sera as assumed by direct EpCAM chromatography compared to the HE2 control group.
  • Pre-serum (Day 1) and immune serum (Day 71) of the multi-epitope vaccine (P2 / 01, HE-2-sialylTn) group compared to the P6 / 01 control group were tested for the immune response against the sialylTn carbohydrate antigen by a sialylTn ELISA.
  • the bicistronic pIRES expression vector from Clontech Laboratories Inc., Palo Alto, USA allows two genes to be expressed at a high level and allows the translation of two consecutive open reading frames from the messenger RNA.
  • the internal ribosome entry site (IRES) was truncated in this expression vector, allowing for lower expression rates in this second reading frame.
  • the attenuated IRES sequence was used for the expression of our selection markers.
  • the DNA manipulations were performed according to standard procedures. Using the PCR technology and Advantage-HF PCR kit (CLONTECH laboratories Inc., Palo Alto, USA), the heavy and light chain of HE2 antibody was amplified. First, primer sequences were used to first introduce the desired restriction sites necessary for incorporation of the gene into the expression vectors, and second, KOZAK sequences were inserted upstream of the open reading frames.
  • the autologous signal sequences were used to direct the naked polypeptide chains into the secretory circulation.
  • the primers were purchased from MWG-Biotech AG, Germany.
  • a two-step cloning technology was developed: the kappa chain containing its autologous signal sequence was amplified as Xho I, Mlu I fragment and ligated into the expression vector using the rapid ligation kit (Roche, Germany) according to the manufacturer's instructions.
  • a chemically competent E. coli bacterial strain DH5alpha (Gibco BRL) was transfected with the construct and amplified using an ampicillin selection marker.
  • the reconstructed IRES sequence and the gamma chain, including the autologous signal sequence were amplified as Mlu I , Nco I and Nco I , Sal I fragments and in a single-step ligation reaction into the modified expression vector already containing the HE2 kappa Chain deboned, ligated.
  • This construct was amplified using E. coli bacterial strain DH5alpha (Gibco BRL). 25 constructs derived from different PCR samples were digested with the restriction endonucleases EcoRI and BamHI. Those constructs showing the correct restriction pattern were bi-directionally sequenced. In this expression construct, the selection cassette described below was installed.
  • the selection marker DHFR was amplified as PCR Xba I / Not I fragment from the pSV2-dhfr plasmid (ATCC # 37146). PCR primers introduced these restriction sites. The attenuated IRES at. Sequence was amplified by PCR from pSV-IRES (Clontech # 6028-1) as Sal I / Xba I fragment. In a single step ligation reaction, IRES at. And DHFR were ligated into the previously described expression construct after digestion with the appropriate restriction endonucleases and another dephosphorylation step.
  • the characterized eukaryotic strain, CHO (ATCC-CRL9096), was transfected with the expression vector described above.
  • the DHFR selection marker was used to establish stable cell lines expressing rHE-2.
  • the cell line was set at cell densities of 10 5 cells in 2 ml complete Iscove's modified Dulbecco's medium with 4 mM L-glutamine to the content of 1.5 g / L Sodium bicarbonate and mixed with 0.1 mM hypoxanthine and 0.016 mM thymidine, 90%; fetal calf serum, seeded 10% (Gibco.BRL).
  • the cells were grown to a 50% cell density.
  • the cells were then transfected according to the instructions of the manufacturers in the absence of serum 2 ug DNA using the Lipofectin ® reagent (Gibco-BRL). The transfection was stopped by addition of complete medium after 6 or 24 hours.
  • the MTX concentration was doubled at each round of selection to a final concentration of 1280 ⁇ M MTX and in parallel was subcultured in 96-well cell culture plates.
  • the supernatants were tested weekly by a specific sandwich ELISA which recognizes both the variable and the constant domain of the antibody. Stable, highest productivity cultures were transferred to 75-cm 2 cell culture flasks and progressively transferred to non-selective medium in 860-cm 2 rolling cell culture flasks. The supernatants were harvested, centrifuged, analyzed and run for further purification.
  • the supernatants were tested with a specific sandwich ELISA recognizing both the variable and the constant domain of the antibody.
  • the polyclonal, anti-idiotypic antibodies IGN111 was coated with a concentration of 10 ug / ml on Maxisorp ® (NUNC) adsorbent sheets. The antibody was raised in goats immunized with HE2 fragments and extracted by affinity by a two-step chromatography procedure. Antibodies to murine constant regions were first adsorbed onto a mouse polyclonal IgG column, and anti-idiotypic antibodies were captured in a second step by affinity on a column of HE2 agarose. The final product, the polyclonal IGN111 antibody preparation, therefore recognizes the variable domain of the HE2 antibody. The remaining active groups were incubated with
  • Isoelectric focusing gels were used to compare the purified expression products with the characterized murine HE2 standard hybridoma antibody.
  • the samples were loaded on IEF gels, pH 3-7 (Invitrogen) and separation performed according to the manufacturer's instructions.
  • the proteins were visualized by silver staining or immunological methods by Western blotting.
  • the proteins were loaded into a Tris buffered SDS / urea / iodoactamide buffer and transferred to nitrocellulose membranes. This was done by the same method as described for Western Blots. Detection was by polyclonal goat IGN111 anti-idiotypic antibody.
  • the interaction of the expression product with the target antigen, EpCAM was analyzed by incubating the purified supernatants with nitrocellulose membranes on the rEpCAM.
  • the staining of the interacting antibodies was carried out in analogy to the Western blot, with an anti-blot.
  • Mouse IgG2a-HRP conjugated antibody (Zymed) was used.
  • a Pharmacia (Amersham Pharmacia Biotech) ⁇ KTA system was used. 1000 ml of clear culture supernatant containing the antibody was concentrated with a Pro-Varion 30 kDa cut-off (Millipore) concentrator, then diluted with PBS and applied to a 20 ml IGN111 Sepharose affinity gel XK26 / 20 column (Amersham Pharmacia Biotech). Contaminating proteins were removed by a wash with PBS + 200 mM NaCl. The bound antibodies were eluted with 100 mM glycine, pH 2.9 and immediately neutralized with 0.5 M NaHCO 3 . The effluent was observed online at ⁇ 215 and ⁇ 280 nm and subjected to a subsequent HPLC analysis on a ZORBAX G-250 (Agilent-technologies) column.
  • the murine HE-2 standard hybridoma antibody recognizes monomeric 25 kDa rEpCAM and also a series of rEpCAM aggregates corresponding to di, tri, and polymeric forms. Exactly the same band distribution was obtained with all purified expression products.
  • the purified expression products and the murine HE-2 standard hybridoma antibody were further investigated. All antibodies show an inhomogeneous poly-band isoelectric focusing pattern, identical in pH, but different in their distribution. They consist of 3 major protein isoforms and two subforms distributed over a pH range of 8.2 to 7.2. CHO-derived isoforms were shifted to higher pH values, the murine HE2 standard shows the identical isoforms, but the quantitative distribution tends to acidic forms.
  • the recombinant mouse IgG2a antibody HE2 could be expressed in CHO cells. Stable genomic integration occurred 14 days after transfection.
  • the expression construct allows rapid and convenient transfection with a single plasmid.
  • the copy number of a plasmid with the corresponding gene and a strong antagonist of this enzyme can be increased by continuously increasing selection pressure.
  • very small amounts of the MTX antagonist can be used for the selection strategy. Moderate expression was achieved with amounts of 10 ⁇ g / 24 h.ml, which can be left in the production cultures for at least 5 weeks.
  • the murine IgG2a antibody 17-1A (17-1A) produced by hybridoma technology was purchased from Glaxo as a 10 mg / ml PBS solution called Panorex®. This antibody was used as murine standard HE2 hybridoma antibody.
  • Recombinant HE-2 was prepared as described above.
  • UPC10 an IgG2a antibody of completely different specificity, was purchased from Sigma (# M9144-1).
  • the vaccine solutions were formulated in 1% Al (OH) 3 suspensions containing 500 ⁇ g antibody / dose.
  • the antibody solutions were tested for endotoxin content by the LAL endpoint method. 10 and 100 ⁇ l supernatants of the solution were tested according to the manufacturer's instructions and compared with an endotoxin standard of 0.15 to 1.2 EU / ml.
  • Antibody solutions were dialyzed against the formulation buffer 1 mM NaH 2 PO 4 , 0.89% NaCl, pH 6.0 using a Slide-A-Lyzer dialysis cassette 3500 MWCO, 3-15 ml (PIERCE, # 0066110). The concentration and integrity of the protein was tested by SEC-HPLC (Zorbax GF250, Agilent).
  • rhesus monkeys monkeys per group with body weights between 4 and 6 kg and without pretreatment were given 500 ⁇ l / animal s.c. vaccinated on day 1, 15, 29 and 57. Serum samples were collected on days 11, 5 and 1 (pre-serum), day 14, day 29, day 57 and day 71.
  • Blood specimens for serum preparation were collected in tubes with coagulation activator and centrifuged 1500g for 30 minutes (according to instructions for use). The serum samples were transferred to tubes and stored at -80 ° C.
  • Preseren and immune sera were analyzed by an ELISA test system with an immunization agent for the testing of the induced immune response.
  • 17-1A was used as coating antibody at a concentration of 10 ⁇ g / ml on Maxisorp® (NUNC) sorption plates diluted with "coating buffer” (PAA, Lot: T05121-436).
  • the remaining active groups were blocked by incubation with 3% FCS (Gibco BRL, heat-inactivated, # 06Q6116K) in PBS before the sera were applied in 6 x 1:10 dilutions in PBS supplemented with 2% FCS.
  • the induced antibodies were detected by their constant regions with a rabbit anti-human IgG, A, M-HRP conjugate (Zymed).
  • the staining was carried out by conventional methods.
  • the absorbance at 492 nm was measured at 620 nm as a reference.
  • the quantification was performed by comparison with standard immune sera containing a standardized amount of antibody comparable to an antibody titer of 9000.
  • the contaminating proteins were removed by a wash with PBS + 200 mM NaCl.
  • the bound antibodies were eluted with 100 mM glycine, pH 2.9 and immediately neutralized with 0.5 M NaHCO 3 .
  • the effluent was measured online at ⁇ 215 and ⁇ 280 nm.
  • the eluted fractions were then subjected to HPLC analysis for IgG / IgM ratio determination, purity and concentration.
  • IgG2a vaccinated animals formed an IgG type immune response that recognizes EpCAM with 30-40% of the immunization-specific antigen titer.
  • the vaccination with IgG2a antibodies therefore led to a cross-reactivity of the immune serum with EpCAM.
  • Deglycosylation of the immunizing antigen significantly reduced both induced IgG levels, both those directed against the immunizing antigen and those against EpCAM.
  • Example 11 Expression of a hybrid immunogenic antibody
  • the recombinant IgG2a Le-Y antibody is an IgG2a hybrid antibody for primate vaccination. It combines the anti-idiotypic Lewis Y (Le-Y) imitating ("mimicking") hypervariable region and the highly immunogenic mouse IgG2a constant regions.
  • Recombinant IgG2a Le-Y antibody immunotherapy enhances the immunogenicity of the original IGN301 antibody produced by a hybridoma cell. It induces a strong immune response against Le-Y and / or EpCAM overexpressed or presented by epithelial tumor cells. This immune response leads to lysis of the tumor cells by complement activation or to the prevention of cell-mediated metastasis.
  • the recombinant IgG2a Le-Y antibody was transiently expressed in HEK293 cells, followed by calcium phosphate co-precipitation in a micro spin system in the presence of FCS. After purification using an anti-Le-Y affinity column and qualification of the expression product, the recombinant IgG2a Le-Y antibody was formulated on Al (OH) 3 and used as a vaccine in rhesus monkey immunization studies with four 500 ⁇ g doses.
  • the IgG-type induced immune response was analyzed by ELISA showing immunization antigen, Le-Y and EpCAM specificity.

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Abstract

L'invention concerne un anticorps immunogène comportant au moins deux épitopes différents d'un antigène associé à une tumeur.

Claims (23)

  1. Anticorps anti-idotypique immunogène, qui présente au moins deux épitopes différents provenant d'antigènes associés à une tumeur, un épitope provenant du groupe des peptides ou des protéines et un épitope provenant du groupe des glucides, l'anticorps reconnaissant l'idiotype d'un anticorps contre un antigène associé à une tumeur.
  2. Anticorps selon la revendication 1, caractérisé en ce qu'il présente au moins un épitope d'un antigène choisi dans le groupe des peptides ou des protéines, en particulier des peptides EpCAM, NCAM, CEA et de lymphocytes T, des glucides, en particulier Lewis Y, SialylTn, Globo H, et des glycolipides, en particulier GD2, GD3 et GM2.
  3. Anticorps selon l'une des revendications 1 ou 2, caractérisé en ce qu'il présente au moins deux épitopes EpCAM.
  4. Anticorps selon l'une des revendications 1 à 3, caractérisé en ce qu'il est conjugué à un peptide, un glycopeptide, un glucide, un lipide ou un acide nucléique.
  5. Anticorps selon la revendication 4, caractérisé en ce que le peptide, le glycopeptide, le glucide, le lipide ou l'acide nucléique représente un épitope d'un antigène associé à une tumeur.
  6. Anticorps selon l'une des revendications 1 à 5, caractérisé en ce qu'il présente au moins un épitope d'EpCAM et au moins un épitope de Lewis Y.
  7. Anticorps selon l'une des revendications 1 à 5, caractérisé en ce qu'il présente au moins un épitope d'EpCAM et au moins un épitope de SialylTn.
  8. Anticorps selon l'une des revendications 1 à 7, caractérisé en ce qu'il est un anticorps humain, humanisé, chimérique ou murin.
  9. Anticorps selon l'une des revendications 1 à 8, caractérisé en ce qu'il est un anticorps recombinant.
  10. Anticorps selon l'une des revendications 1 à 9, caractérisé en ce qu'il est un dérivé d'anticorps choisi dans le groupe comprenant des fragments d'anticorps, des conjugués d'anticorps ou des homologues d'anticorps.
  11. Anticorps selon l'une des revendications 1 à 10, caractérisé en ce que l'antigène de l'idiotype est choisi dans le groupe des peptides ou protéines en particulier, des peptides EpCAM, NCAM, CEA et de lymphocytes T.
  12. Anticorps selon l'une des revendications 1 à 11, caractérisé en ce que l'antigène de l'idiotype est choisi dans le groupe des glucides, en particulier Lewis Y, SialylTn, Globo H, et des glycolipides, en particulier GD2, GD3 et GM2.
  13. Préparation pharmaceutique contenant un anticorps immunogène selon l'une des revendications 1 à 12.
  14. Agent diagnostique contenant un anticorps immunogène selon l'une des revendications 1 à 12.
  15. Formulation vaccinale contenant un anticorps immunogène selon l'une des revendications 1 à 12.
  16. Formulation vaccinale selon la revendication 15, caractérisée en ce que l'anticorps est contenu dans une quantité immunogène de 0,01 µg à 10 mg.
  17. Formulation vaccinale selon l'une des revendications 15 ou 16, caractérisée en ce qu'au moins un adjuvant vaccinal est contenu.
  18. Procédé de production d'un anticorps immunogène selon l'une des revendications 1 à 12 par
    a) la mise à disposition d'un anticorps présentant l'idiotype d'un antigène associé à une tumeur et
    b) le couplage d'au moins un épitopc d'un antigène associé à une tumeur ou son imitation à un anticorps.
  19. Procédé de production d'un anticorps immunogène selon l'une des revendications 1 à 12, par
    a) la mise à disposition d'un acide nucléique codant pour un anticorps présentant l'idiotype d'un antigène associé à une tumeur et
    b) la recombinaison de l'acide nucléique avec un acide nucléique qui code pour un épitope d'un antigène associé à une tumeur ou son imitation.
  20. Procédé de production d'un anticorps immunogène selon la revendication 1, caractérisé en ce qu'un épitope d'un antigène associé à une tumeur ou son imitation est conjugué à l'anticorps comme support.
  21. Procédé selon la revendication 20, caractérisé en ce que l'antigène est choisi dans le groupe des peptides ou protéines, en particulier des peptides EpCAM, NCAM, CEA et de lymphocytes T, de glucides, en particulier Lewis Y, SialylTn, Globo H, et les glycolipides, en particulier GD2, GD3 et GM2.
  22. Procédé selon la revendication 20 ou 21, caractérisé en ce qu'un acide nucléique codant pour un épitope d'un antigène de peptide ou de protéine est conjugué à l'anticorps.
  23. Procédé selon l'une des revendications 20 à 22, caractérisé en ce que l'anticorps présente un autre épitope d'un antigène associé à une tumeur.
EP03726990A 2002-05-15 2003-05-15 Anticorps monoclonal immunogene Expired - Lifetime EP1503799B1 (fr)

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AT0074402A AT502293B1 (de) 2002-05-15 2002-05-15 Immunogener, monoklonaler antikörper
AT7442002 2002-05-15
PCT/AT2003/000142 WO2003097663A2 (fr) 2002-05-15 2003-05-15 Anticorps monoclonal immunogene

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EP1503799A2 EP1503799A2 (fr) 2005-02-09
EP1503799B1 true EP1503799B1 (fr) 2011-11-30

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AT500650B1 (de) 2003-04-17 2009-11-15 Altropus Gmbh Immunogener rekombinanter antikörper
AT504160A1 (de) * 2006-09-11 2008-03-15 Ralf Dr Kircheis Verwendung einer mehrkomponenten-tumorvakzine

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US6010902A (en) * 1988-04-04 2000-01-04 Bristol-Meyers Squibb Company Antibody heteroconjugates and bispecific antibodies for use in regulation of lymphocyte activity
WO1993010763A1 (fr) * 1991-11-26 1993-06-10 Jenner Technologies Vaccins antitumoraux
UA40577C2 (uk) * 1993-08-02 2001-08-15 Мерк Патент Гмбх Біспецифічна молекула, що використовується для лізису пухлинних клітин, спосіб її одержання, моноклональне антитіло (варіанти), фармацевтичний препарат, фармацевтичний набір (варіанти), спосіб видалення пухлинних клітин
EP0805628B1 (fr) * 1995-01-17 2003-05-02 Brigham And Women's Hospital, Inc. Transport transepithelial specifique de recepteurs d'immunogenes
WO1997040140A1 (fr) * 1996-04-22 1997-10-30 The Wistar Institute Of Anatomy And Biology Anticorps anti-idiotypiques contre gd2 et leurs applications
US6676946B2 (en) * 1997-03-27 2004-01-13 Institut Pasteur Multiple antigen glycopeptide carbohydrate vaccine comprising the same and use thereof
HU226150B1 (en) * 1999-01-13 2008-05-28 Igeneon Krebs Immuntherapie Use of antibodies for anticancer vaccination
EP1178785B1 (fr) * 1999-05-06 2008-12-24 Wake Forest University Compositions et procedes d'identification d'antigenes provoquant une reponse immunitaire
AT409086B (de) * 1999-11-16 2002-05-27 Igeneon Krebs Immuntherapie Neue verwendung von antikörpern als impfstoffe
MXPA02008247A (es) * 2000-03-21 2004-04-05 Igeneon Krebs Immuntherapie Conjugado de polisacarido-polipeptido.
GB0102145D0 (en) * 2001-01-26 2001-03-14 Scancell Ltd Substances
AU2002308562B2 (en) * 2001-05-03 2008-01-24 Merck Patent Gmbh Recombinant tumor specific antibody and use thereof

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ES2380659T3 (es) 2012-05-17
EP1503799A2 (fr) 2005-02-09
AU2003232907A8 (en) 2003-12-02
US20100310551A1 (en) 2010-12-09
AT502293A1 (de) 2007-02-15
AU2003232907A1 (en) 2003-12-02
DK1503799T3 (da) 2012-03-19
WO2003097663A3 (fr) 2004-03-18
AT502293B1 (de) 2008-03-15
ATE535259T1 (de) 2011-12-15
WO2003097663A2 (fr) 2003-11-27
US20050181475A1 (en) 2005-08-18

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